Identification and Optimization of Pyrrolo [3, 2-d] pyrimidine Toll-like

d]pyrimidine Toll-like Receptor 7 (TLR7) Selective. Agonists for the Treatment of Hepatitis B. David C. McGowan,*. ,†. Florence Herschke,*. ,†. Fr...
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Identification and Optimization of Pyrrolo[3,2‑d]pyrimidine Toll-like Receptor 7 (TLR7) Selective Agonists for the Treatment of Hepatitis B David C. McGowan,*,† Florence Herschke,*,† Frederik Pauwels,† Bart Stoops,† Ilham Smyej,† Stefaan Last,† Serge Pieters,† Werner Embrechts,† Mourad Daoubi Khamlichi,§ Tine Thoné,† Bertrand Van Schoubroeck,† Wendy Mostmans,† Debbie Wuyts,† Dorien Verstappen,† Annick Scholliers,† Dorien De Pooter,† Deborah Dhuyvetter,† Herman Borghys,† Marianne Tuefferd,† Eric Arnoult,‡ Jin Hong,∥ Gregory Fanning,† Jacques Bollekens,† Vijay Urmaliya,† Ard Teisman,† Helen Horton,† Tim H. M. Jonckers,† and Pierre Raboisson† †

Janssen Pharmaceutica, N. V. Turnhoutseweg 30, 2340 Beerse, Belgium Janssen Research & Development L.L.C., 1400 McKean Road, Spring House, Pennsylvania 19454, United States § Villapharma Research S.L., Parque Tecnológico de Fuente Á lamo, Ctra. El Estrecho-Lobosillo, Km. 2.5-Av. Azul, 30320 Fuente Á lamo de Murcia, Murcia, Spain ∥ Alios Biopharma, Inc., 260 East Grand Avenue, South San Francisco, California 94080, United States ‡

S Supporting Information *

ABSTRACT: Pyrrolo[3,2-d]pyrimidines were identified as a new series of potent and selective TLR7 agonists. Compounds were optimized for their activity and selectivity over TLR8. This presents an advantage over recently described scaffolds that have residual TLR8 activity, which may be detrimental to the tolerability of the candidate drug. Oral administration of the lead compound 54 effectively induced a transient interferon stimulated gene (ISG) response in mice and cynomolgus monkeys. We aimed for a high first pass effect, limiting cytokine induction systemically, and demonstrated the potential for the immunotherapy of viral hepatitis.



INTRODUCTION Chronic hepatitis B affects over 350 million people worldwide, as it may eventually lead to cirrhosis or hepatocellular carcinoma (HCC), responsible for one million deaths every year.1,2 Less than 1 in 10 patients achieves functional cure, defined as HBsAg loss and sustained HBV-DNA suppression with or without the appearance of HBsAb (seroconversion), with the approved treatments, nucleosides/nucleotides, and interferon-α (IFNα).3−5 Low cure rates are due to high viral antigen loads, inability of the host immune system to control HBV, and the immunosuppressive environment of the liver.6,7 IFNα therapy, unlike nucleosides/nucleotides, has the potential to control all three issues when directly exposed to hepatocytes and liver nonparenchymal cells. IFNα treatment of HBV-infected hepatocytes, which induces the expression of interferon-stimulated genes (ISG),8 decreases both HBsAg and HBeAg levels.9 IFNα also induces the maturation of myeloid dendritic cells toward a Th1 immune response, which is key to controlling HBV.10 The major reason for the limited clinical use is the intramuscular route of administration, leading to local and systemic exposure of exogenous recombinant IFNα responsible for both injection site and systemic side effects.10 © 2017 American Chemical Society

Conversely, orally administered agents inducing endogenous IFNα, like TLR7 agonists, could be more potent and better tolerated.11 Toll-like receptors (TLRs) play an important role in human innate immunity by recognizing pathogen associated molecular patterns (PAMPs) present in a variety of viruses and microorganisms.12−14 TLR7 recognizes foreign genetic material in the form of single-stranded viral RNA. The receptor is localized in the endosome of plasmacytoid dendritic cells (pDC) and B lymphocytes.15,16 Detection of foreign nucleic acid in pDCs leads to an antiviral immune response, resulting in an abundant production of endogenous IFNα and leading to the transcription of interferon stimulated genes (ISGs).8,15,16 In B cells, activation of TLR7 induces B cell maturation and secretion of antibodies which could promote HBsAg seroconversion.17,18 TLR8 shares a similar protein structure, is also involved in the detection of viral RNA, but differs in that it is found to be expressed on myeloid dendritic cells and monocytes rather than on pDC and B cells.19 TLR8 agonism may have the advantages of a strong Th1 response but may Received: March 9, 2017 Published: July 3, 2017 6137

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Figure 1. Described TLR 7 agonists and novel pyrrolo[3,2-d]pyrimidine (9).

Scheme 1. Representative Synthetic Route of Pyrrolo[3,2-d]pyrimidinesa

a Reagents and conditions: (a) BnBr, Cs2CO3, DMF, 70 °C, 50% isolated yield; (b) n-butylamine, 1,4-dioxane, 100 °C, 67% yield; (c) HOAc, aq HCl, 100 °C, 24 h, 40%; (d) NaN3, NMP/H2O 9/1, microwave 170 °C, 5 h, 86%; (e) PPh3, 1,4-dioxane:H2O 9:1, 120 °C, 16 h, 76%.

Scheme 2. Representative Alternative Synthesis of Pyrrolo[3,2-d]pyrimidinesa

a

Reagents and conditions: (a) 1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea, HOAc, NaOCH3, CH3OH, rt, 16 h, 87%; (b) POCl3, DIPEA, ACN, 70 °C, 1 h, 46%; (c) 2-pyridylmethanol, PPh3, DIAD, THF, rt, 30 min, 65%; (d) NaOH, 1,4-dioxane, 60 °C, 5 h, 75%; (e) 5-methyl-2oxazolemethanamine, DMSO, 110 °C, 16 h, 15%; (f) (5-methyloxazol-2-yl)methanol), NaH, THF, 0−50 °C, 30 min, 22%. See patent applications WO2014056953 and WO2014207082.

kg resulted in a Cmax that was 2-fold lower than that achieved following iv infusion of 0.1 mg/kg 3. However, mean IFNα plasma levels induced following oral dosing were greater than 60-fold higher than that produced by iv infusion.23Drug candidates having a rapid elimination after oral administration would potentially improve the overall tolerability and limit the risk for tachyphylaxis, and this was indeed one objective in this program. Another objective was to maximize the selectivity of the TLR7 agonist series over TLR8, to limit the induction of pro-inflammatory cytokines (e.g., TNFα). Indeed, 3 has been reported to be 30-fold selective for human TLR7 vs TLR8.24 A structural observation of known TLR 7 agonists from Figure 1 reveals several common aspects. They all contain, at minimum, a bicyclic core scaffold, with an aryl group pendant, or fused as in imiquimod. Contained in the core structure is a

suffer drawbacks such as the induction of proinflammatory cytokines.20 This manuscript focuses on the effort toward an oral, small-molecule TLR7 selective agonist with a high first pass effect that would limit IFNα levels in the plasma, resulting in a presystemic immune modifying response that could be beneficial toward the treatment of chronic HBV. There are several TLR7 agonist series described to date; some examples are shown in Figure 1. Imiquimod (1) is a marketed medication to treat actinic keratosis and basal cell carcinoma.22 A second example of TLR7 selective agonist from the literature is 2, of the 8-hydroxyadenine series, described as a potent IFN inducer in both mice and monkeys.21The pteridone series of agonists, represented by GS-9620 (3), was examined in clinical trials to treat HBV.11 A cynomolgus monkey study performed with 3 has demonstrated that an oral dose of 2 mg/ 6138

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Table 1. Structure−Activity Relationship of Benzyl Group Replacementsa

a All compounds had a CC50 > 24 μM in the HEK 293 cytotoxicity assay (see Supporting Information). bLEC; least effective concentration values were averaged when determined in two or more independent experiments. See Supporting Information.

atoms, connected by an oxygen or nitrogen atom and positioned three to four bonds away from the aryl group. If indeed there is a recognition element of the TLR7 protein

minimal pattern of a nitrogen hydrogen bond acceptor and an adjacent, unsubstituted NH2 hydrogen bond donor. A third common aspect is the hydrophobic tail, typically four carbon 6139

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in a modest 67% yield. On the side, the 2-chlorine was converted to a hydroxyl group by heating in aqueous acetic acid to afford 7. Finally, nucleophilic aromatic substitution at the 2chlorine with sodium azide in a mixture of NMP/water afforded the 2-azido intermediate 8, followed by a Staudinger reduction that provided the initial agonist 9 after purification via silica gel column chromatography.28 For later analogues, the synthesis was ameliorated to a more scalable route, avoiding the hazardous use of azides and high temperature microwave reaction.29 Scheme 2 depicts an example of the synthetic pathway starting with the condensation between ethyl 3-amino-1H-pyrrole-2-carboxylate and 1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea, analogous to methods described in the literature.30 The resulting 4-hydroxypyrrolo[3,2-d]pyrimidyl carbamate (10) was converted to the 4-chloropyrrolo[3,2-d]pyrimidinyl carbamate (11) via phosphorus oxychloride in 46% yield. Alkylation of the pyrrole nitrogen under standard Mitsunobu conditions proceeded in moderate yield (65%) to afford 12. This was followed by base mediated deprotection of the 2amino carbamate (13) and finally displacement of the chlorine by an amine nucleophile in a polar solvent that provided 37 or by an oxygen nucleophile, as in the third subseries, facilitated by NaH (e.g., 55).

toward RNA bases, then perhaps new scaffolds would best imitate the shape and electrostatics of adenine and guanine. The design of pyrrolo[3,2-d]pyrimidine (9) started from the concept to use the core pyrimidine scaffold of previously described TLR agonists,25,26keeping the hydrogen bond donor and acceptor pattern of the pyrimidine and adding a fused ring so that the pendent benzyl group was placed proximal to the alkyl chain. Among the many permutations attempted in our laboratory, we would like to focus this manuscript on the success of the TLR7 selective pyrrolo[3,2-d]pyrimidine series starting from the initial compound synthesized (9). Indeed, this novel structure holds all three of the desired design attributes. The butyl chain and pendent benzyl group project ipsilaterally from the bicyclic aromatic scaffold containing a similar donor− acceptor pattern as the described agonists in Figure 1. We employed two in vitro assays to assess the activity of compounds during the SAR exploration. In the first in vitro assay, the ability of compounds to agonize human TLR7 and TLR8 was assessed in a cell-based reporter assay using HEK293 cells transiently transfected with a wild-type TLR7 or TLR8 expression vector and a NFκB-luc reporter construct. To confirm the activity, compounds were tested in the second in vitro assay, using IFN-mediated anti-HCV activity in the replicon system upon incubation with conditioned media from human peripheral blood mononuclear cells (hPBMCs). In this assay, activation of human TLR7 on pDCs results in the production of IFN. Promising derivatives were evaluated in rat pharmacokinetic studies and a mouse pharmacokinetic− pharmacodynamic model. The selected lead candidate was tested in the mouse hydrodynamic injection (HDI) model and finally in cynomolgus monkey.



RESULTS AND DISCUSSION Structure−Activity Relationship. Changes in the 2-amino group led to inactivity (e.g., 7 and 8 were >25 μM [hTLR7, 8]) and so further modifications were not pursued. The first example, having an unsubstituted phenyl ring and an n-butyl carbon chain (9), showed exceptional TLR7 activity but unsatisfactory selectivity over the hERG receptor (IC50 = 2.1 μM). When the phenyl group was more distal to the scaffold (e.g., 14, 15), TLR7 potency resulted in the same range as 9; however undesired TLR8 activity was observed. Replacement of the phenyl group of 9 with an amide led to lower activity (16) but improved with the isobutene replacement (17). The thiophene analogue 18 had TLR7 selectivity superior to 9 in contrast to the 2-thiazole congener 19 with a selectivity ratio of only 12. The imidazo[1,2-a]pyridine fragment (20) and the three pyridine analogs 21−23 demonstrated that TLR7 activity was gained when the nitrogen of the heterocycle was adjacent to the methylene group. This substitution pattern showed that the pyridine ring could be modified (24−26) and still retain TLR7 selectivity with the exception of the 2-pyridyl-6-methoxy analogue (27) that exhibited outstanding potency on TLR7 but carried undesired TLR8 activity. Indeed, 21 displayed remarkable activity and selectivity and lower CYP450 inhibition than the phenyl (9) or thiophene (18) congeners (see Supporting Information) and would be held constant for exploration in the following subseries. In addition, extensive possibilities exist to access substituted pyridines, facilitating future SAR exploration. The subsequent subseries of analogues would focus on systematic changes of the amine group. Variation of the amine while holding the unsubstituted pyridyl group constant is described in Table 2. Differences in activity were seen when the butyl chain was replaced by other aliphatic or heteroaromatic groups; all were selective for TLR7 over TLR8. An ether variation on the butyl chain of 21 displayed a loss of activity (28). The same held true for alkyl ethers 29 and 30. A modest decline in activity for amino alcohol 31 was observed when compared to 21 but highlighted that a polar group was well



MEDICINAL CHEMISTRY STRATEGY Our strategy was to systematically investigate the structure− activity relationship of this novel series of pyrrolo[3,2d]pyrimidines, which was divided into three subseries. The initial subseries, represented by analogues 9−27 (Table 1), where the n-butylamine was conserved and the phenyl ring is replaced by aliphatic and heterocyclic substituents, was designed to optimize activity and selectivity toward TLR7. The second subseries (compounds 28−45) allowed the exploration of alternatives to the aliphatic amine. Finally, in the third subseries (compounds 46−59), to afford selective TLR7 agonists with limited off-target activity, the nitrogen atom of 42 was replaced with an oxygen that afforded the lead compound 54 for further evaluation. In the context of this project and in contrast to a traditional medicinal chemistry program, rapidly metabolized compounds were desired, thus limiting compound exposure and systemic cytokine activation. Furthermore, it has been demonstrated that a robust antiviral response can be achieved in non-human primates with less than daily dosing of a TLR7 agonist.27 Despite the expected low compound plasma concentrations, an effort was made to remove off-target activity including, for example, the potential to inhibit the hERG potassium ion channel.



CHEMISTRY The synthesis of this series of pyrrolo[3,2-d]pyrimidines is depicted in Scheme 1 and began with 2,4-dichloropyrrolo[3,2d]pyrimidine (4). Alkylation with benzyl bromide in DMF afforded N-benzyl analogue 5 in 50% yield. Displacement of the first chlorine atom with n-butylamine in 1,4-dioxane afforded 6 6140

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0.2 vs 1.2 μM, respectively). Finally, if the N−O bond in the isoxazole was reversed (45), then potency was lost. Compound 42 had achieved the potency and selectivity that was desired and was identified to have no appreciable receptor kinase activity, and no CYP inhibition across several isozymes (see Supporting Information). However, 42 remained a strong hERG binder (IC50 = 1.2 μM). The strategy to identify the best lead candidate was 2-fold: First, the preferred 5-methylisoxazole moiety was held constant allowing the examination of the effect of substituted pyridines, which could influence hERG receptor binding as suggested in the literature.31Second, it has been described that introduction of an oxygen atom into an aliphatic chain can reduce the propensity of hERG binding.32 Thus, we explored pyridine ring variations and converted the amine connection of the heterocycle (Table 3, R5) to an ether (Table 3, X = O). Variations in the pyridine substitution (compounds 46−53) afforded several potent agonists, especially when substituted by electron rich moieties (46, 47). It is described in the literature that within a given series of compounds there can be a correlation between hERG binding and a measurement of lipophilicity such as cLogP.33 However, within this chemical series, there was a weak correlation between cLogP and hERG IC50. On the contrary, the best progress in avoiding hERG inhibition came with the discrete structural modification of 42 to the corresponding ether 54, despite the two compounds having nearly the same cLogP of 1.1. This modification led to an improved cardiovascular safety profile with a minor loss in TLR7 potency. Further heterocyclic ether variants (oxazole 55, thiazole 56, oxadiazole 57) also bore relatively no hERG inhibition and had intriguing differences in potency that may be rationalized by their difference in charge distribution. The electrostatic potential alignment of analogs 54−57 showed that the most potent compounds share a similar electrostatic profile (Figure 2; see Supporting Information). There are two positions on the ring on either side of the methylene group that are key. One α position (south side, Figure 2) can be electronegatively charged, while the other (north side) must be electropositively charged (thiazole) or neutral as the potency decreases when this side of the molecule becomes strongly electronegative, as in oxadiazole derivative 57. The electrostatic profile of the best substituent could be used as a signature for further exploration of this series. Finally, substituted pyridines 58 and 59 once again displayed that potency could be regained by the addition of electron donating methoxy groups on the pyridine ring but unfortunately afforded decreased selectivity over the hERG antitarget. The binding mode of 54, obtained by docking into monkey TLR7 (see Supporting Information), illustrated in Figure 2, corresponds to the expected binding mode and is comparable to the binding mode described for Resiquimod.34 Strong, direct dual hydrogen bonds between the 2-amino group of 54, the acidic side chain of Asp555, and the backbone of Thr586 (Carbonyl) were observed. An additional H-bond between the 3-position of the pyrimidine and the backbone of Thr586 (amino) contributes to the stabilization of the compound in the binding site. The scaffold is further stabilized by a π-stacking interaction with Phe408 on one side and by CH−π interactions with Leu557 on the other side. The methylisoxazole is deeply buried in the hydrophobic pocket formed by residues Phe349, Phe351, Val381, and Ile585, stabilized by a π-stacking interaction with Phe351, and has good

Table 2. Structure−Activity Relationship of Amine Substitutiona

a All compounds were >25 μM LEC [hTLR8] and showed CC50 > 24 μM in the HEK 293 cytotoxicity assay described in the Supporting Information. bLEC; least effective concentration values were averaged when determined in two or more independent experiments. See Supporting Information.

tolerated. Heterocyclic substituents presented a range of activity depending on the ring size and position of the heteroatoms. The bis-pyridyl analogues (32, 33) did not show an increase in potency over the butyl chain, and furthermore the racemic αmethyl group of 33 did not display a favorable increase in activity, and so further modifications were not pursued. In contrast, thiazole and 1,3,4-oxadiazole (34, 35) ring modifications were deleterious to activity, suggesting that the electrostatic properties of the ring are important in addition to the regiochemisty of the heterocycle. The oxazoles displayed higher potency when the methyl group was placed next to the oxygen (37), as opposed to neighboring the nitrogen atom (38). Both oxazoles showed binding to the serotonin receptors (e.g., IC50(5HT2a) = 0.65 and 2.3 μM, respectively). The methylisoxazole 42 was an equipotent TLR7 agonist and had less inhibition of the 5HT2a receptor (IC50 = 6.2 μM). The cyclopropyl congener (43) was also an effective agonist but displayed more potent hERG inhibition than 42 (hERG IC50 = 6141

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Table 3. Modifications to the Heterocyclic Ringsa

All compounds were >25 μM LEC [hTLR8] and showed CC50 > 24 μM in the HEK293 cytotoxicity assay described in the Supporting Information. n.d., not done. bLEC values were averaged when determined in two or more independent experiments.

a

Figure 2. Left: Electrostatic potential comparison, based on AM1-BCC charges, between isoxazole (54), oxazole (55), thiazole (56), and oxadiazole (57). Right: Predicted binding mode by docking (Glide XP) for compound 54 in a monkeyTLR7 structure (one TLR7 monomer in cyan and the other in green). See Supporting Information. 6142

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lists the in vitro metabolic stability of 54 in liver microsomes and whole-cell hepatocytes across species. Intrinsic clearance (CLint) in liver microsomes was moderate in mouse, dog, and human and high in rat and monkey. The intrinsic clearance in hepatocytes evaluated the potential for non-CYP mediated metabolism and was low to moderate in mouse, dog, and human and high in monkey and rat. Plasma protein binding across species remained relatively low and ranged from 54% to 63% bound. 54 had limited inhibition of the hERG potassium ion channel 3H-dofetilide binding in vitro (IC50 > 50 μM), which correlated with a relatively low functional hERG inhibition in the QPatch planar patch clamp assay (32% inhibition at 3 μM).35 To evaluate whether this effect, albeit low, or other undetected compound characteristics could lead to undesired electrophysiology effects in vivo, 54 was subjected to a cardiovascular safety pharmacology study in anesthetized female guinea pigs. In this model, electrophysiological changes were measured after administration of 54 by iv infusion in pentobarbital anesthetized animals (cumulatively dosed up to 4.9 mg/kg iv). From this study, no major cardioelectrophysiological effects were observed up to maximal total plasma exposures of 9700 ng/mL (at the end of the infusion). There was no tendency for the formation of micronuclei in vitro in the presence and absence of rat S9 fraction and no potential for mutagenicity revealed by the AMES II test. In further support of the lead compound, cytokine induction was measured after compound stimulation of whole blood of healthy volunteers ex vivo and included IFNα, IFNγ, IL-12p40, and IL-12p70. Pharmacokinetics. Among preclinical species, the in vitro metabolism in rat hepatocytes was exceptionally high, and the in vivo clearance was predicted to be above rat liver blood flow. A pharmacokinetic study in healthy male Sprague-Dawley rats investigated the exposure of 54 in systemic circulation following intravenous administration at 2.5 mg/kg and in the portal vein, liver, and systemic circulation following oral administration of 10 mg/kg. The iv clearance correlated well with the in vitro clearance and exceeded rat liver blood flow, resulting in a very short terminal half-life. The maximum concentration in the portal vein was equal to 314 ng/mL and occurred 1 h postdose. The concentration measurements in the liver and systemic circulation were below quantifiable limits. Oral administration in dogs (1 mg/kg) resulted in fast absorption (Tmax at 0.5 h, first sampled time point). The Cmax was 125 ng/mL and the systemic concentration dropped below 10 ng/mL, 4 h after administration. The systemic concentration in dog was higher than in rat which is in line with the in vitro stability in microsomes and hepatocytes. Pharmacokinetic and Pharmacodynamic Studies in Mice and Cynomolgus Monkeys. A mouse in vivo model was used to demonstrate the initial proof of concept to induce endogenous IFNα. Single oral administration of 0.3, 1, 3, and 10 mg/kg doses of 54 was given to healthy, female, fasted C57Bl/6 mice. Concentrations of compound and mouse-IFN via ELISA were measured from the plasma and compared to vehicle. The results are shown in Table 6. 54 was found to be rapidly cleared in conjunction with our target profile. Both Cmax and AUC increased less than dose proportionally between 0.3 and 3 mg/kg and more than dose-proportionally between 3 and 10 mg/kg. Tmax occurred between 0.5 and 1 h, and a liver to plasma ratio of 2 was measured. Levels of IFNα peaked between 1 and 2 h after administration in both plasma and liver. Increase in IFNα was dose-proportional in plasma but sigmoidal in the liver, suggesting a possible saturation of

surface complementarities. In addition, the pyridine ring of 54 has a CH−π interaction with the Cα of Gln354. In comparison, the binding mode of 54 is less favorable in hTLR8 for two main reasons (illustrated in Figure S1): (1) Leu557 is replaced by Asp545 in hTLR8. The scaffold no longer has a CH−π interaction. Instead, Asp545 is making nonfavorable electrostatic interactions with the compound. (2) Thr586 is replaced by Thr574 in hTLR8. The side chain of Thr574 adopts a different orientation, providing less room to accommodate 54. The Thr574 side chain is making only one H-bond with the 2-amino group instead of two with its backbone in monkey TLR7. In addition, the Phe351 (TLR7) is replaced by Tyr358 in TLR8 and does not allot enough room to accommodate the methylisoxazole in the hydrophobic pocket. The combination of both a less stable interaction of the scaffold and the methylisoxazole predicts that 54 would be less potent on hTLR8. Agonist 54 was found to be selective for TLR7 over TLR8 in the same cellular reporter assay using HEK293 cells transiently transfected with mouse, rat, or cynomolgus TLR7 or TLR8 expression vector and NFκB-luciferase reporter construct (see Supporting Information). In addition, no direct activation of NFκB was observed. After repetition at higher concentration, 54 was found to be very selective over TLR8 (>100 μM LEC human TLR8) (Table 4). Table 4. Activity of 54 across Species in Vitroa transient transfection of HEK293 cells with TLR7 + NFκB-luc

TLR8 + NFκB-luc

species

average LEC (μM)

human mouse rat cynomolgus monkey human mouse rat cynomolgus monkey

0.41 0.34 0.33 0.80 >100 >25 >25 >25

NFκB-luc a

>25

See Supporting Information for n and confidence interval.

The permeability of 54 was measured in a MDCK-MDR1 cellular assay at 5 μM. 54 displayed decent permeability with a Papp (A → B, in the presence of GF120918) of 41 × 106 cm s−1 and an efflux ratio of 18, indicating that the compound is a P-gp substrate. 54 demonstrated low inhibition across five CYP450 isozymes (IC50 > 10 μM; see Supporting Information) and was also not a time dependent inhibitor of CYP450 3A4. Table 5 Table 5. In Vitro Metabolic Stability and Plasma Protein Binding of 54a species mouse rat dog cynomolgus monkey human a

CLint microsomes ((μL/min)/mg)

CLint hepatocytes ((μL/min)/106 cells)

plasma protein binding (% bound)

55 >346 44 87

19 >277 7 80

63 61 62 52

25

6

54

Values were rounded to the nearest whole number. 6143

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model. This model allows an initial assessment of the anti-HBV activity, as mice develop an acute infection that is cleared within 2 weeks.36 54 was administered orally to C57Bl/6 male mice 1 day after HDI for 5 consecutive days at 0.3, 1, 2.5, and 5 mg/kg q.d. and was found to be well tolerated (no clinical observations were noted). The dose was chosen based on the robust ISG upregulation observed in healthy C57Bl/6 mice (Table 6). 54 induced a 2.7 log decrease in serum HBV viral load from 0.3 mg/kg, and a maximum 3.1 log decrease was observed for doses between 1 and 5 mg/kg (Figure 3). Also, 1 day after the last dose, mice were sacrificed to evaluate liver HBV viral load (Figure 3). A small but statistically significant (one-way ANOVA with Dunnett’s multiple comparison) decrease was observed at all doses and was maximal for the 5 mg/kg dose (0.63 log decrease). In contrast, a similar decrease in viral load, in either the serum or the liver, required a higher dose of 3 (20 mg/kg). Healthy cynomolgus monkeys were chosen as the next preclinical pharmacokinetic−pharmacodynamic model, as they are the closest available model to human immunology (Table 7).37 Oral doses of 1, 3, and 10 mg/kg of 54 were well tolerated (no clinical observations and no relevant change in body weight, body temperature observed). Maximum mean plasma concentration occurred at 0.25−2 h postdose and increased dose-proportionally, despite the large variability observed. The low oral bioavailability (F < 1%) can potentially be attributed to a high first pass effect, assuming that absorption was similar to that generated in the rat hepatic portal vein pharmacokinetic study. According to the mode of action, a transient and dosedependent decrease in lymphocyte counts and increase in neutrophil counts were observed from 1 mg/kg.38−40 Cytokines were measured in plasma by Luminex or ELISA. IFNα production was detected in all animals starting from the 3 mg/kg dose and showed a more than dose-proportional profile of maximum response, despite high interindividual variability. Interestingly, an ISG response was induced starting from 1 mg/ kg: Isg15, Oas1b, Oasl, Mx1, Cxcl11, If it44l where upregulation (measured by microarray) was detected from 1 mg/kg and peaked at 4−8 h postdose. A second wave of gene induction was observed at 72 h postdose, including ISG Cxcl10, as well as monocyte-induced Ccl18. Monocyte activation was also suggested by the production of MCP1 (from 1 mg/kg) and

Table 6. Mean Maximum Levels in the Plasma and Liver and ISG Upregulation, Induced by 54 after Oral Dosing in C57Bl/6 Micea dose (mg/kg) Cmax plasma (ng/mL) Cmax liver (ng/g) IFNαmax plasma (pg/mL) IFNαmax liver (pg/g) Oas1bmax plasmab Oas1bmax liverb Isg15max plasmab Isg15max liverb Mx1max plasmab Mx1max liverb Tlr7max plasmab Tlr7max liverb

0.3

1

3

10

3.7 6.8 105 BQL 1.85 16 1.35 10 12 60 3.6 3.15

7.1 6.3 205 55 2.1 14 2 60 18 315 3.7 2.45

2.8 23.0 560 470 2.45 11 1.95 75 20 745 4.05 2.9

72.9 152.3 1365 580 2.2 14 1.75 100 19 775 4.15 3.7

a

Groups of nine mice were dosed with 0.3, 1, 3, or 10 mg/kg of 54. At 0.5 h, blood was drawn to quantify compound. At 1, 2, or 4 h, three mice of each group were sacrificed and blood was drawn to measure plasma and liver compound levels (by LC−MS), IFNα (by ELISA) and ISG expression levels (by RT-qPCR). Values were rounded to the nearest whole number. BQL, below quantifiable limit. bFold-change relative to vehicle.

endogenous IFNα production especially in the liver (Table 6). ISG (interferon-stimulated gene) expression was measured in parallel by RT-qPCR. Interestingly, all three Isg15, Oas1b, and Mx1 were upregulated to a higher level in the liver than in the plasma, while IFNα, Cxcl10, and the target Tlr7 were upregulated similarly in blood and liver (Table 6). These data reveal that 54 can induce an antiviral ISG response without inducing an IFNα response at a low dose. One potential drawback of the TLR7 selective agonist 2 is the significant plasma concentrations observed at 8 h in mice after an oral dose of 1 mg/kg,22 in contrast to 54 that was below quantifiable limits at the same dose and time point. Therefore, compound 54 has rapid clearance after oral administration in mice, limiting compound exposure and systemic cytokine activation, resulting in an improved potential tolerability profile. The potential of 54 to inhibit HBV replication in vivo was tested in the hydrodynamic injection (HDI) HBV mouse

Figure 3. Efficacy of 54 in the HDI-HBV mouse model. Left: Serum HBV viral load over time (mean ± SD). Right: Liver HBV viral load at end of dosing (qPCR, mean ± SD). Seven groups of eight male C57Bl/6 mice, 4−6 weeks old, were administered 20 μg of pAAV2-HBV1.3mer genotype D plasmid by hydrodynamic injection. One day later, groups 1−7 received compound 54 orally at 0, 0.3, 1, 2.5, and 5 mg/kg q.d., respectively, for 5 days. HPbCD20% at acidic pH was used as vehicle. At different time points, blood was sampled to measure serum HBV viral load by qPCR. One day after the last dose, mice were euthanized to measure liver HBV viral load by qPCR. LLOQ indicates lower limit of quantification. 6144

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Plasma Protein Binding. The free fraction in mouse, rat, dog, cyno, and human plasma was determined by rapid equilibrium dialysis (RED device, Thermo Fisher Scientific, Geel, Belgium). The RED device consists of a 48-well plate containing disposable inserts bisected by a semipermeable membrane creating two chambers. A 300 μL aliquot of plasma containing test compound at 5 μM was placed on one side and 500 μL of phosphate buffered saline (PBS) on the other. The plate was sealed and incubated at 37 °C for 4.5 h. Samples were removed from both the plasma and buffer compartment and analyzed for test compound using a specific LC−MS/MS method to estimate free and bound concentrations. Permeability and Efflux. The in vitro permeability and potential to be transported by P-glycoprotein (P-gp) was determined using an MDCK cell line transfected with human MDR1 (P-glycoprotein). 5 μM 54 was added to the apical (A) or basolateral (B) side of a confluent monolayer of MDCK-MDR1 cells. Apparent permeability in the A → B direction in absence (Papp(A→B) = 5.5 × 10−6 cm/s) and presence (Papp(A→B) = 41 × 10−6 cm/s) of GF120918 (P-gp inhibitor) and in the B → A direction in absence of GF120918 (Papp(A→B) = 23 × 10−6 cm/s) was measured by monitoring the appearance of the test compound on the opposite side of the membrane using a specific LC−MS/MS method. The efflux ratios ((B → A, GF120918)/(A → B, GF120918)) were calculated to determine whether the test compound was a P-gp substrate. All in vivo studies were performed in AAALAC-accredited sites, and ethical approval by the corresponding ethical committee was obtained. Chemistry. Reagents and solvents were purchased from commercial sources and used without purification. All final products were >95% pure. NMR spectra were recorded on a Bruker Avance 400 spectrometer, operating at 400 MHz for 1H NMR and 101 MHz for 13 C NMR, or a Bruker 360 spectrometer, operating at 360 MHz for 1H NMR and 91 MHz for 13C NMR. Chemical shifts and multiplicity data are given according to the ACS NMR guidelines. High resolution mass spectrometry was performed on a Waters Acquity IClass UPLC-DAD and Xevo G2-S QTOF. The samples were run on a Waters BEH C18 (1.7 μm, 2.1 mm × 50 mm, at 50 °C) column with a flow rate of 1 mL/min, using reverse phase chromatography with a gradient from 95% A to 5% A in 4.6 min and holding for 0.4 min (A, 95% CH3COONH4 6.5 mM + 5% CH3CN; B, CH3CN). 5-Benzyl-2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine (5). Into a 50 mL vial were placed 2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine (1 g, 5.32 mmol), DMF (10 mL), DIPEA (2.75 mL, 16 mmol), and benzyl bromide (0.7 mL, 5.85 mmol). The vial was sealed and shaken for 16 h at rt. The reaction mixture was filtered through silica, and the solvent of the filtrate was removed under reduced pressure. The crude was used without further purification in the next step. 5-Benzyl-N-butyl-2-chloro-5H-pyrrolo[3,2-d]pyrimidin-4amine (6). Into a 50 mL round-bottom flask equipped with a magnetic stir bar were placed 5-benzyl-2,4-dichloro-5H-pyrrolo[3,2d]pyrimidine (1.4 g, 5.03 mmol), n-butylamine (0.59 mL, 6.04 mmol), and 1,4-dioxane (5 mL). The flask was equipped with a reflux condenser and allowed to heat with stirring at 100 °C for 16 h. After cooling to rt, the solvents were removed under reduced pressure. The crude was purified via silica gel column chromatography using a heptane to EtOAc gradient. The best fractions were pooled and the solvents were removed under reduced pressure to afford the title compound. 5-Benzyl-4-(butylamino)-5H-pyrrolo[3,2-d]pyrimidin-2-ol (7). A suspension of 6 (70 mg, 0.22 mmol) in acetic acid (1 mL) and conc HCl (0.2 mL) was heated in a sealed tube at 100 °C for 24 h. The solvent was removed under reduced pressure, and the crude was purified via silica gel column chromatography using a gradient of CH2Cl2 to CH2Cl2/CH3OH (95:5). 1H NMR (300 MHz, DMSO-d6) δ 10.33 (s, 1H), 7.16−7.35 (m, 4H), 6.98 (d, J = 6.74 Hz, 2H), 6.19 (t, J = 5.43 Hz, 1H), 5.85 (d, J = 2.75 Hz, 1H), 5.52 (s, 2H), 3.22−3.30 (m, 2H), 1.23−1.37 (m, 2H), 0.91−1.06 (m, 2H), 0.73−0.80 (m, 3H). 2-Azido-5-benzyl-N-butyl-5H-pyrrolo[3,2-d]pyrimidin-4amine Hydrochloride (8). Into a glass vial equipped with a magnetic stir bar were placed 5-benzyl-N-butyl-2-chloro-5H-pyrrolo[3,2-d]pyrimidin-4-amine (1 g, 3.18 mmol), NaN3 (0.62 g, 9.53 mmol),

Table 7. Mean Plasma Pharmacokinetic and Pharmacodynamic Parameters of 54 in Cynomolgus Monkey after Administration by Oral Gavagea dose (mg/kg) Cmax (ng/mL) Tmax (h) AUC0−last (ng·h/mL) IFNα maxa MCP1 maxa IL-1Ra maxa IL-6 maxa G-CSF maxa IL-10 maxa

1

3

10

4.12 1−2 NA BQL 325 (2/5) 75 (5/5) 20 (4/5) 90 (3/5) 20 (3/5)

11.2 0.5−2 26.7 120 (3/5) 1560 5/5) 1820 (5/5) 10 (3/5) 75 (2/5) 45 (3/5)

23.4 0.5−2 57.5 2980 (5/5) 8295 (5/5) 10575 (5/5) 525 (5/5) 120 (4/5) 125 (5/5)

a

pg/mL, frequency; BQL, below quantification limit; NA, not applicable. Five cynomolgus monkeys were administered 1, 3, or 10 mg/kg of 54 by oral gavage. At 0 (predose), 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 h postdose, blood was sampled to measure compound concentrations in plasma, cytokine levels (by ELISA or Luminex), and gene expression (by microarray). Doses were administered in different animals but staggered by 3 days for acute toxicity evaluation.

IL-1Ra (from 3 mg/kg). General proinflammatory cytokines IL-6 and G-CSF were measurable from 10 mg/kg, while antiinflammatory cytokine IL-10 was induced from 3 mg/kg. This is encouraging in regard to the safety of compound 54. Overall, these results demonstrate that 54 is more than simply an interferon inducer and warrant further evaluation for HBV indications.



CONCLUSION In this manuscript, the research on a novel class of TLR7 selective agonists for immunotherapy of hepatitis B was described. This new series of pyrrolo[3,2-d]pyrimidines was designed by taking key elements of chemical series previously described in the literature and combining them into a new structure. We found 54 to be potent and selective over other TLRs in preclinical species. Oral administration of 54 was well tolerated in vivo, displayed a high first pass effect, and induced an interferon stimulated gene response in both mice and cynomolgus monkeys. This ISG response was of higher magnitude in the liver, the site of viral infection, than in the periphery in mice. In the HDI-HBV mouse model, 54 reduced viral load in the serum and in the liver. In cynomolgus monkey, 54 did not induce IFNα systemically after a low (1 mg/kg) oral dose but induced an upregulation of ISGs. In summary, we have identified a series of novel and potent TLR7 agonists and presented data that indicate the potential for the immunotherapy of viral hepatitis.



EXPERIMENTAL SECTION

Intrinsic Clearance (CLint) of 54. CLint was determined in mouse, rat, dog, cynomolgus monkey, and human liver microsomes. Incubations were performed at 37 °C at a compound concentration of 1 μM and a microsomal protein concentration of 0.5 mg/mL. Samples were removed at intervals of up to 60 min and analyzed for the concentration of compound to determine its intrinsic clearance rate. 54 was incubated in mouse, rat, dog, cynomolgus monkey, and human hepatocytes (106 cells/mL) at 1 μM for 0, 10, 20, 40, 60, and 120 min. Samples were removed at intervals of up to 120 min and analyzed for the concentration of compound to determine its intrinsic clearance rate. 6145

DOI: 10.1021/acs.jmedchem.7b00365 J. Med. Chem. 2017, 60, 6137−6151

Journal of Medicinal Chemistry

Article

and NMP/water (9:1, 4 mL). The glass vial was sealed, and the mixture was heated with stirring to 170 °C for 5 h. After cooling to rt, the mixture was diluted with EtOAc (20 mL) and washed with water (5 × 15 mL). The organic layer was dried over magnesium sulfate, the solids were removed via filtration, and the solvents of the filtrate were removed under reduced pressure. The crude was purified via silica gel column chromatography using a heptane to EtOAc gradient. The best fractions were combined and the solvents were removed under reduced pressure. The product was suspended in 0.5 mL of dioxane, and to it was added 1 equiv of 4 N HCl in dioxane at 0 °C. The mixture was stirred for 16 h at room temperature and then the solvent was removed under reduced pressure to afford the title compound. LC−MS (M + H) m/z = 322. 1H NMR (300 MHz, chloroform-d) δ 7.32−7.42 (m, 3H), 7.18−7.22 (m, 2H), 6.99−7.08 (m, 2H), 6.90 (d, J = 3.16 Hz, 1H), 5.51 (s, 2H), 4.95 (br s, 1H), 3.37 (dt, J = 5.22, 6.94 Hz, 2H), 1.13−1.26 (m, 2H), 0.98 (qd, J = 7.31, 14.90 Hz, 2H), 0.69− 0.78 (m, 3H). 5-Benzyl-N4-butyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (9). Into a glass vial equipped with a magnetic stir bar were placed 2azido-5-benzyl-N-butyl-5H-pyrrolo[3,2-d]pyrimidin-4-amine (100 mg, 0.311 mmol), 1,4-dioxane (4 mL), water (1 mL), and triphenylphosphine (245 mg, 0.93 mmol). The vial was sealed and the mixture heated with stirring to 120 °C for 16 h. After cooling to rt, the solvents were removed under reduced pressure. The crude was purified via silica gel column chromatography using a CH2Cl2 to 10% CH3OH in CH2Cl2 gradient. The best fractions were pooled and the solvents were removed under reduced pressure to afford the title compound. 1H NMR (400 MHz, DMSO-d6) δ 7.25−7.33 (m, 3H), 7.19−7.25 (m, 1H), 6.95−7.01 (m, 2H), 5.98 (d, J = 2.86 Hz, 1H), 5.73 (t, J = 5.50 Hz, 1H), 5.49 (s, 2H), 5.23 (s, 2H), 3.25−3.32 (m, 2H), 1.28−1.39 (m, 2H), 1.04 (qd, J = 7.39, 15.02 Hz, 2H), 0.73−0.82 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 158.8, 151.7, 150.8, 139.4, 133.2, 129.0, 127.8, 126.5, 110.4, 99.7, 52.0, 39.8, 31.5, 19.8, 14.2. ESI-HRMS (TOF) m/z: 296.1878 [M + H]+ (calcd for C17H21N5, 295.1797). Methyl (4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-2-yl)carbamate (10). Amino-2-ethoxycarbonylpyrrole hydrochloride (25.8 g, 135.7 mmol) was partitioned between CH2Cl2 and sat. aq NaHCO3, the organic layer was dried over MgSO4, the solids were removed by filtration, and the filtrate was evaporated to dryness. The residue was dissolved in CH3OH (500 mL) along with 1,3bis(methoxycarbonyl)-2-methyl-2-thiopseudourea (32.1 g, 156 mmol) and acetic acid (39 mL, 619 mmol) and stirred at rt for 1 h. A precipitate appeared, and stirring was continued overnight. NaOCH3 (73.1 g, 1353 mmol) was added. An exotherm was observed, and the reaction mixture was stirred overnight. The reaction mixture was brought to pH 5 via addition of acetic acid and the precipitate was isolated by filtration, triturated with water (2 × 350 mL), CH3CN (350 mL), and diisopropyl ether (350 mL). Methyl (4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-2-yl)carbamate (11). Methyl N-(4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin2-yl)carbamate (25 g, 120 mmol) was dispensed into acetonitrile (350 mL) in a 500 mL multineck flask at rt. POCl3 (22.1 mL, 238.2 mmol) was added, and the reaction mixture was heated to 70 °C while stirring by an overhead, mechanical stirrer at 300 rpm. N,N-Diisopropylethylamine (41.4 mL, 240.2 mmol) was added dropwise by a syringe pump at a flow rate of 0.2 mL/min. The reaction mixture was cooled to rt and poured into a stirred solution of sodium acetate (78.8 g, 961 mmol) in water (500 mL) at 45 °C. The organic solvents were removed under reduced pressure, and the remaining liquid was stirred and cooled in an ice bath. The formed solid was isolated by filtration, washed with CH3CN, and triturated with diisopropyl ether to afford the title compound as an off-white solid. 1H NMR (400 MHz, DMSOd6) 12.27 (br s, 1H), 10.22 (s, 1H), 7.92 (t, J = 2.97 Hz, 1H), 6.57 (dd, J = 1.76, 3.08 Hz, 1H), 3.66 (s, 3H). Methyl (4-Chloro-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidin-2-yl)carbamate (12). To a suspension of methyl (4chloro-5H-pyrrolo[3,2-d]pyrimidin-2-yl)carbamate (5 g, 22 mmol), 2pyridinemethanol (2.6 mL, 26.5 mmol), and polystyrene-bound triphenylphosphine (18.4 g, 55.2 mmol) in anhydrous THF (153 mL) was added DIAD (6.9 mL, 33 mmol) at rt, and the reaction

mixture was stirred for 30 min, then was concentrated under reduced pressure. The product was purified via silica gel column chromatography using a gradient of CH2Cl2/CH3OH 100:0 to 90:10. The product fractions were collected and concentrated under reduced pressure. The product was recrystallized in CH3CN, isolated by filtration, and dried under vacuum to afford the title compound as a white solid. 4-Chloro-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidin2-amine (13). Methyl (4-chloro-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidin-2-yl)carbamate (4.5 g, 14.2 mmol) was dissolved in 1,4-dioxane (68 mL) in a 100 mL round-bottom flask, and NaOH (1 N aq, 34 mL) was added. The mixture was heated at 60 °C for 5 h. The mixture was cooled and concentrated under reduced pressure. The crude was treated with water and the precipitate was isolated by filtration and dried to afford the title compound. The product was used without further purification in the next step. N4-Butyl-5-(2-phenoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine2,4-diamine (14). 1H NMR (360 MHz, DMSO-d6) δ 7.20−7.28 (m, 3H), 6.92 (t, J = 7.50 Hz, 1H), 6.82 (dd, J = 0.73, 8.78 Hz, 2H), 6.32 (t, J = 5.31 Hz, 1H), 5.94 (d, J = 2.93 Hz, 1H), 5.36 (br s, 2H), 4.62 (t, J = 5.31 Hz, 2H), 4.16 (t, J = 5.12 Hz, 2H), 3.39−3.49 (m, 2H), 1.58 (quin, J = 7.32 Hz, 2H), 1.35 (qd, J = 7.38, 14.82 Hz, 2H), 0.89 (t, J = 7.32 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.7, 156.6, 149.3, 148.9, 131.2, 128.3, 119.7, 113.1, 108.9, 97.6, 66.7, 46.5, 38.4, 30.1, 18.6, 12.6. ESI-HRMS (TOF) m/z: 326.1980 (calcd for C18H23N5O [M + H]+, 326.1980). N4-Butyl-5-(3-phenylpropyl)-5H-pyrrolo[3,2-d]pyrimidine2,4-diamine (15). 1H NMR (400 MHz, DMSO-d6) δ 7.22−7.30 (m, 2H), 7.08−7.22 (m, 4H), 6.34 (br t, J = 5.50 Hz, 1H), 5.94 (d, J = 3.08 Hz, 1H), 5.59 (br s, 2H), 4.29 (t, J = 7.04 Hz, 2H), 3.38−3.51 (m, 2H), 2.39−2.55 (m, 2H), 1.82−1.99 (m, 2H), 1.49−1.64 (m, 2H), 1.34 (qd, J = 7.40, 14.99 Hz, 2H), 0.90 (t, J = 7.37 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 157.6, 151.1, 148.8, 141.6, 132.8, 128.8, 128.5, 126.3, 109.9, 98.3, 48.7, 40.1, 33.8, 32.3, 31.8, 20.2, 14.3. ESIHRMS (TOF) m/z: 324.2189 (calcd for C19H25N5 [M + H]+, 324.2188). 2-(2-Amino-4-(butylamino)-5H-pyrrolo[3,2-d]pyrimidin-5yl)-N-methylacetamide (16). 1H NMR (400 MHz, DMSO-d6) δ 8.32 (br d, J = 4.40 Hz, 1H), 7.11 (d, J = 3.08 Hz, 1H), 6.93 (t, J = 5.17 Hz, 1H), 5.94 (d, J = 2.86 Hz, 1H), 5.38 (br s, 2H), 4.74 (s, 2H), 3.31−3.45 (m, 2H), 2.64 (d, J = 4.62 Hz, 3H), 1.49−1.64 (m, 2H), 1.29−1.44 (m, 2H), 0.92 (t, J = 7.37 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.4, 158.5, 151.4, 150.1, 132.7, 111.4, 99.5, 51.9, 31.6, 26.1, 20.2, 14.3. ESI-HRMS (TOF) m/z: 277.1779 (calcd for C13H20N6O [M + H]+, 277.1777). N 4 -Butyl-5-(3-methylbut-2-en-1-yl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (17). 1H NMR (360 MHz, DMSO-d6) δ 7.14 (d, J = 2.93 Hz, 1H), 5.98 (t, J = 5.49 Hz, 1H), 5.89 (d, J = 2.93 Hz, 1H), 5.41 (br s, 2H), 5.21 (ddd, J = 1.46, 5.21, 6.50 Hz, 1H), 4.83 (br d, J = 6.59 Hz, 2H), 3.34−3.47 (m, 2H), 1.73 (s, 3H), 1.69 (s, 3H), 1.48−1.61 (m, 2H), 1.34 (qd, J = 7.35, 14.91 Hz, 2H), 0.87−0.96 (m, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.5, 149.4, 148.5, 134.0, 130.2, 120.2, 108.7, 97.1, 45.4, 38.3, 30.0, 24.0, 18.4, 16.6, 12.6. ESIHRMS (TOF) m/z: 274.2034 (calcd for C15H23N5 [M + H]+, 274.2031) N 4 -Butyl-5-(thiophen-3-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine Hydrochloride (18). 1H NMR (400 MHz, DMSO-d6) δ 12.68 (br s, 1H), 7.55−7.67 (m, 2H), 7.50 (dd, J = 2.86, 4.84 Hz, 1H), 7.37 (br s, 2H), 7.25 (br s, 1H), 6.82 (d, J = 4.84 Hz, 1H), 6.22 (d, J = 2.86 Hz, 1H), 5.70 (s, 2H), 3.48 (q, J = 6.53 Hz, 2H), 1.44 (quin, J = 7.15 Hz, 2H), 1.11 (qd, J = 7.29, 14.88 Hz, 2H), 0.75−0.90 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 153.1, 151.9, 138.8, 136.7, 134.8, 127.7, 126.7, 122.8, 108.5, 95.4, 48.0, 40.4, 31.1, 19.7, 14.2. ESI-HRMS (TOF) m/z: 302.1442 (calcd for C15H19N5S [M + H]+, 302.1439). N 4 -Butyl-5-(thiazol-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (19). 1H NMR (360 MHz, DMSO-d6) δ 7.77 (d, J = 3.29 Hz, 1H), 7.67 (d, J = 3.29 Hz, 1H), 7.36 (d, J = 2.93 Hz, 1H), 6.50 (t, J = 5.31 Hz, 1H), 5.99 (d, J = 3.29 Hz, 1H), 5.80 (s, 2H), 5.33 (s, 2H), 3.28−3.40 (m, 2H), 1.49 (quin, J = 7.23 Hz, 2H), 6146

DOI: 10.1021/acs.jmedchem.7b00365 J. Med. Chem. 2017, 60, 6137−6151

Journal of Medicinal Chemistry

Article

1.24 (qd, J = 7.49, 14.87 Hz, 2H), 0.86 (t, J = 7.32 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 166.6, 157.2, 150.0, 149.2, 141.0, 130.9, 120.2, 108.8, 98.9, 47.7, 38.3, 29.8, 18.3, 12.6. ESI-HRMS (TOF) m/z: 303.1393 (calcd for C14H18N6S [M + H]+, 303.1392) N4-Butyl-5-(imidazo[1,2-a]pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (20). 1H NMR (400 MHz, DMSOd6) δ 8.49−8.60 (m, 1H), 7.83−7.92 (m, 2H), 7.50 (dd, J = 0.66, 9.02 Hz, 1H), 7.39 (d, J = 2.86 Hz, 1H), 7.29 (ddd, J = 1.10, 6.71, 9.13 Hz, 1H), 6.92 (dt, J = 1.10, 6.71 Hz, 1H), 5.97 (d, J = 2.86 Hz, 1H), 5.69 (br s, 2H), 5.49 (s, 2H), 3.41−3.50 (m, 2H), 1.60 (quin, J = 7.26 Hz, 2H), 1.31 (qd, J = 7.40, 14.99 Hz, 2H), 0.86 (t, J = 7.37 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 157.3, 151.6, 144.5, 143.2, 132.6, 127.8, 126.2, 116.7, 113.0, 111.4, 110.7, 98.4, 46.5, 40.5, 31.4, 20.2, 14.2. ESI-HRMS (TOF) m/z: 336.1941 (calcd for C18H21N7 [M + H]+, 336.1937). N 4 -Butyl-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (21). 1H NMR (360 MHz, DMSO-d6) δ 8.54 (d, J = 4.76 Hz, 1H), 8.07 (br t, J = 5.12 Hz, 1H), 7.85 (dt, J = 1.46, 7.68 Hz, 1H), 7.57 (d, J = 2.93 Hz, 1H), 7.38 (dd, J = 5.12, 7.32 Hz, 1H), 7.34 (d, J = 7.68 Hz, 1H), 6.56 (br s, 2H), 6.11 (d, J = 2.93 Hz, 1H), 5.57 (s, 2H), 3.40−3.51 (m, 2H), 1.54 (quin, J = 7.23 Hz, 2H), 1.15−1.31 (m, 2H), 0.87 (t, J = 7.32 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.8, 156.2, 149.6, 148.7, 147.5, 136.7, 130.8, 122.1, 121.7, 121.1, 109.1, 97.9, 52.2, 29.8, 18.4, 12.6. ESI-HRMS (TOF) m/z: 297.1833 (calcd for C16H20N6 [M + H]+, 297.1828). N 4 -Butyl-5-(pyridin-3-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (22). 1H NMR (360 MHz, DMSO-d6) δ 8.44 (dd, J = 1.46, 4.76 Hz, 1H), 8.25 (d, J = 1.83 Hz, 1H), 7.49 (d, J = 2.93 Hz, 1H), 7.27−7.36 (m, 1H), 7.22−7.27 (m, 1H), 6.75 (t, J = 5.27 Hz, 1H), 6.21 (br s, 2H), 6.12 (d, J = 2.93 Hz, 1H), 5.65 (s, 2H), 3.33−3.40 (m, 2H), 1.28−1.42 (m, 2H), 0.91−1.06 (m, 2H), 0.71− 0.80 (m, 3H). 13C NMR (91 MHz, DMSO-d6) δ 154.8, 150.2, 147.5, 149.8, 146.4, 132.8, 132.6, 132.5, 122.5, 107.9, 96.8, 48.0, 39.3, 29.8, 18.1, 12.6. ESI-HRMS (TOF) m/z: 297.1833 (calcd for C16H20N6 [M + H]+, 297.1828). N 4 -Butyl-5-(pyridin-4-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (23). 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 5.16 Hz, 2H), 7.39 (d, J = 3.08 Hz, 1H), 6.83 (d, J = 5.94 Hz, 2H), 6.19 (br s, 1H), 6.08 (d, J = 2.86 Hz, 1H), 5.66−5.75 (m, 2H), 5.60 (s, 2H), 3.17 (br. s., 2H), 1.32 (td, J = 7.54, 14.86 Hz, 2H), 0.97 (sxt, J = 7.39 Hz, 2H), 0.69−0.79 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 157.7, 151.1, 150.2, 148.3, 133.8, 121.3, 110.0, 99.3, 50.9, 40.9, 31.4, 19.7, 14.1. ESI-HRMS (TOF) m/z: 297.1835 (calcd for C16H20N6 [M + H]+, 297.1828). N4-Butyl-5-((6-methylpyridin-2-yl)methyl)-5H-pyrrolo[3,2d]pyrimidine-2,4-diamine (24). 1H NMR (400 MHz, DMSO-d6) δ 7.59−7.83 (m, 2H), 7.46 (br d, J = 2.20 Hz, 1H), 7.23 (br d, J = 7.48 Hz, 1H), 7.07 (br d, J = 7.48 Hz, 1H), 6.02 (br d, J = 2.42 Hz, 1H), 5.81 (br s, 2H), 5.44 (s, 2H), 3.44 (br d, J = 5.50 Hz, 2H), 2.49 (s, 3H), 1.47−1.69 (m, 2H), 1.17−1.39 (m, 2H), 0.89 (br t, J = 7.26 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 157.8, 157.2, 156.9, 151.6, 147.2, 138.8, 132.8, 123.2, 120.0, 110.4, 98.7, 53.8, 31.6, 24.2, 20.1, 14.2. ESI-HRMS (TOF) m/z: 311.1988 [M + H]+ (calcd for C17H22N6: 310.1906). ESI-HRMS (TOF) m/z: 311.1988 (calcd for C17H22N6 [M + H]+, 311.1984). N 4 -Butyl-5-(quinolin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (25). 1H NMR (360 MHz, DMSO-d6) δ 8.39 (d, J = 8.42 Hz, 1H), 7.95−8.02 (m, 2H), 7.83 (ddd, J = 1.46, 6.95, 8.42 Hz, 1H), 7.63 (t, J = 7.39 Hz, 1H), 7.56 (d, J = 3.20 Hz, 1H), 7.26 (d, J = 8.42 Hz, 1H), 7.02 (t, J = 5.31 Hz, 1H), 6.05 (d, J = 2.93 Hz, 1H), 5.70 (s, 2H), 5.53 (br s, 2H), 3.32−3.45 (m, 2H), 1.38− 1.48 (m, 2H), 1.11 (qd, J = 7.46, 14.96 Hz, 2H), 0.71 (t, J = 7.32 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 157.1, 156.5, 149.4, 148.0, 145.2, 136.8, 131.1, 129.0, 126.9, 126.8, 125.9, 125.6, 118.6, 108.9, 97.8, 53.0, 38.3, 29.9, 18.3, 12.4. ESI-HRMS (TOF) m/z: 347.1983 (calcd for C20H22N6 [M + H]+, 347.1984). N4-Butyl-5-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (26). 1H NMR (400 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.86 (t, J = 4.95 Hz, 1H), 7.30 (d, J = 3.08 Hz, 1H), 5.92 (d, J = 2.86 Hz, 1H), 5.44 (s, 2H), 5.23 (s, 2H),

3.71 (s, 3H), 3.39 (dt, J = 5.28, 6.93 Hz, 2H), 2.32 (s, 3H), 2.21 (s, 3H), 1.53−1.69 (m, 2H), 1.37 (qd, J = 7.38, 14.83 Hz, 2H), 0.93 (t, J = 7.37 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 164.8, 158.6, 155.3, 151.2, 150.6, 148.7, 132.6, 126.8, 126.0, 111.3, 99.3, 60.3, 52.1, 40.3, 31.5, 20.3, 14.3, 13.4, 11.1. ESI-HRMS (TOF) m/z: 355.2244 (calcd for C19H26N6O [M + H]+, 355.2246). N4-Butyl-5-((3-methoxypyridin-2-yl)methyl)-5H-pyrrolo[3,2d]pyrimidine-2,4-diamine (27). 1H NMR (360 MHz, DMSO-d6) δ 8.10 (dd, J = 1.46, 4.76 Hz, 1H), 7.61 (t, J = 4.94 Hz, 1H), 7.54 (dd, J = 1.10, 8.42 Hz, 1H), 7.42 (dd, J = 4.76, 8.42 Hz, 1H), 7.21 (d, J = 2.93 Hz, 1H), 5.90 (d, J = 2.93 Hz, 1H), 5.38 (s, 2H), 5.24 (s, 2H), 3.90 (s, 3H), 3.42 (dt, J = 5.12, 6.95 Hz, 2H), 1.59−1.68 (m, 2H), 1.41 (qd, J = 7.30, 15.05 Hz, 2H), 0.91−0.97 (m, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.9, 152.7, 149.3, 148.9, 144.4, 138.4, 131.2, 123.9, 118.4, 109.3, 97.5, 54.6, 47.6, 38.7, 29.8, 18.6, 12.6. ESI-HRMS (TOF) m/z: 327.1934 (calcd for C17H22N6O [M + H]+, 327.1933). N4-(2-Methoxyethyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2d]pyrimidine-2,4-diamine (28). 1H NMR (360 MHz, DMSO-d6) δ 8.55 (dd, J = 0.73, 4.03 Hz, 1H), 7.82 (dt, J = 1.83, 7.68 Hz, 1H), 7.43 (t, J = 5.31 Hz, 1H), 7.40 (d, J = 3.29 Hz, 1H), 7.35 (dd, J = 5.12, 7.32 Hz, 1H), 7.29 (d, J = 8.05 Hz, 1H), 5.96 (d, J = 2.93 Hz, 1H), 5.44 (s, 2H), 5.39 (br s, 2H), 3.56 (q, J = 5.37 Hz, 2H), 3.41−3.52 (m, 2H), 3.24 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.9, 156.0, 149.5, 147.7, 149.2, 136.8, 130.9, 122.1, 121.2, 109.2, 98.0, 69.5, 56.8, 52.2, 38.5. ESI-HRMS (TOF) m/z: 299.1624 (calcd for C15H18N6O [M + H]+, 299.1620). N4-(3-Methoxypropyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (29). 1H NMR (360 MHz, DMSOd6) δ 8.55 (d, J = 4.76 Hz, 1H), 7.81 (dt, J = 1.46, 7.68 Hz, 1H), 7.38 (d, J = 3.26 Hz, 1H), 7.32−7.37 (m, 1H), 7.15 (d, J = 7.87 Hz, 1H), 7.05 (t, J = 5.14 Hz, 1H), 5.97 (d, J = 2.93 Hz, 1H), 5.48 (s, 2H), 5.30 (s, 2H), 3.39−3.43 (m, 2H), 3.31 (t, J = 6.40 Hz, 2H), 3.19 (s, 3H), 1.78 (quin, J = 6.59 Hz, 2H). 13C NMR (91 MHz, DMSO-d6) δ 157.1, 156.4, 149.6, 149.5, 147.6, 136.7, 130.9, 122.0, 121.0, 109.2, 98.2, 68.7, 56.7, 52.2, 36.2, 27.8. ESI-HRMS (TOF) m/z: 313.1780 (calcd for C16H20N6O [M + H]+, 313.1777). N4-(2-Ethoxyethyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (30). 1H NMR (360 MHz, DMSO-d6) δ 8.55 (d, J = 5.12 Hz, 1H), 7.81 (dt, J = 1.83, 7.68 Hz, 1H), 7.39 (d, J = 2.93 Hz, 1H), 7.34 (dt, J = 5.12, 7.14 Hz, 2H), 7.25 (d, J = 7.68 Hz, 1H), 5.96 (d, J = 2.93 Hz, 1H), 5.44 (s, 2H), 5.33 (s, 2H), 3.52−3.58 (m, 2H), 3.47−3.52 (m, 2H), 3.42 (q, J = 7.07 Hz, 2H), 1.07 (t, J = 6.95 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 157.1, 156.2, 149.6, 149.5, 147.7, 136.8, 130.9, 122.1, 121.2, 109.3, 98.2, 67.4, 64.3, 52.2, 46.8, 14.0. ESI-HRMS (TOF) m/z: 313.1781 (calcd for C16H20N6O [M + H]+, 313.1777). 1-((2-Amino-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)amino)butan-2-ol (31). 1H NMR (360 MHz, DMSO-d6) δ 8.54 (d, J = 4.76 Hz, 1H), 7.81 (dt, J = 1.83, 7.68 Hz, 1H), 7.39 (d, J = 2.93 Hz, 1H), 7.34 (dd, J = 4.94, 6.77 Hz, 1H), 7.18− 7.27 (m, 2H), 5.96 (d, J = 2.93 Hz, 1H), 5.47 (s, 2H), 5.30 (s, 2H), 4.84 (br s, 1H), 3.54 (quin, J = 5.85 Hz, 1H), 3.41−3.50 (m, 1H), 3.24−3.34 (m, 1H), 1.21−1.43 (m, 2H), 0.86 (t, J = 7.32 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.9, 156.1, 149.6, 149.5, 147.7, 147.7, 136.6, 130.9, 121.9, 121.0, 98.1, 69.4, 52.1, 44.9, 26.2, 8.8. ESI-HRMS (TOF) m/z: 313.1699 (calcd for C16H20N6O [M + H]+, 313.1777). N4-((5-Methylpyridin-2-yl)methyl)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (32). 1H NMR (360 MHz, DMSO-d6) δ 8.43 (d, J = 4.89 Hz, 1H), 8.32 (s, 1H), 7.79 (dt, J = 1.83, 7.68 Hz, 1H), 7.65 (t, J = 5.67 Hz, 1H), 7.38−7.47 (m, 2H), 7.32 (dd, J = 4.94, 6.77 Hz, 1H), 7.12 (d, J = 8.05 Hz, 1H), 6.95 (d, J = 8.05 Hz, 1H), 6.00 (d, J = 2.93 Hz, 1H), 5.55 (s, 2H), 5.31 (s, 2H), 4.68 (d, J = 5.49 Hz, 2H), 2.25 (s, 3H). 13C NMR (91 MHz, DMSOd6) δ 157.2, 156.5, 155.4, 150.1, 149.3, 147.8, 142.5, 136.7, 135.7, 131.4, 129.6, 122.0, 120.8, 119.3, 109.4, 98.4, 52.3, 43.7, 16.5. ESIHRMS (TOF) m/z: 346.1785 (calcd for C19H19N7 [M + H]+, 346.1780). N4-(1-(Pyridin-2-yl)ethyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (33). 1H NMR (360 MHz, DMSOd6) δ 8.46−8.51 (m, 2H), 7.84 (t, J = 7.56 Hz, 1H), 7.80 (d, J = 7.41 6147

DOI: 10.1021/acs.jmedchem.7b00365 J. Med. Chem. 2017, 60, 6137−6151

Journal of Medicinal Chemistry

Article

Hz, 1H), 7.66 (dt, J = 1.83, 7.68 Hz, 1H), 7.45 (d, J = 2.93 Hz, 1H), 7.25−7.38 (m, 3H), 7.21 (dd, J = 5.12, 7.32 Hz, 1H), 5.96 (d, J = 2.93 Hz, 1H), 5.47−5.58 (m, 2H), 5.42 (t, J = 6.95 Hz, 1H), 5.24 (s, 2H), 1.52 (d, J = 6.95 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 161.9, 156.8, 155.9, 148.4, 147.4, 141.0, 136.7, 136.6, 135.2, 124.8, 122.0, 121.0, 119.7, 119.1, 115.0, 97.9, 52.2, 48.7, 20.2. ESI-HRMS (TOF) m/z: 346.1780 (calcd for C19H19N7 [M + H]+, 346.1780). N4-((2-Methylthiazol-4-yl)methyl)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (34). 1H NMR (360 MHz, DMSO-d6) δ 8.44 (d, J = 4.87 Hz, 1H), 7.75−7.85 (m, 2H), 7.41 (d, J = 2.93 Hz, 1H), 7.33 (dd, J = 4.94, 7.50 Hz, 1H), 7.23 (d, J = 7.32 Hz, 1H), 7.01 (s, 1H), 5.98 (d, J = 2.20 Hz, 1H), 5.49 (s, 2H), 5.36 (s, 2H), 4.67 (d, J = 5.49 Hz, 2H), 2.63 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 163.9, 157.1, 156.3, 153.4, 150.0, 149.2, 147.7, 136.9, 131.2, 122.1, 121.3, 113.1, 109.4, 98.3, 52.2, 17.7. ESI-HRMS (TOF) m/z: 352.1353 (calcd for C17H17N7S [M + H]+, 352.1344). N4-((5-Methyl-1,3,4-oxadiazol-2-yl)methyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (35). 1H NMR (360 MHz, DMSO-d6) δ 8.45 (d, J = 4.76 Hz, 1H), 8.05 (t, J = 5.67 Hz, 1H), 7.80 (dt, J = 1.83, 7.68 Hz, 1H), 7.44 (d, J = 2.93 Hz, 1H), 7.34 (dd, J = 5.12, 6.95 Hz, 1H), 7.23 (d, J = 8.05 Hz, 1H), 5.99 (d, J = 2.93 Hz, 1H), 5.49 (s, 2H), 5.37 (s, 2H), 4.85 (d, J = 5.85 Hz, 2H), 2.43 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 163.5, 162.3, 156.7, 156.0, 150.3, 148.5, 147.5, 136.7, 131.6, 122.0, 121.1, 109.2, 98.2, 52.0, 33.3, 9.2. ESI-HRMS (TOF) m/z: 337.1525 (calcd for C16H16N8O [M + H]+, 337.1525). N4-(Oxazol-2-ylmethyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (36). 1H NMR (360 MHz, DMSOd6) δ 8.42 (d, J = 4.89 Hz, 1H), 8.08 (t, J = 5.67 Hz, 1H), 8.00 (s, 1H), 7.81 (dt, J = 1.83, 7.68 Hz, 1H), 7.43 (d, J = 2.93 Hz, 1H), 7.28−7.36 (m, 2H), 7.14 (s, 1H), 5.98 (d, J = 2.93 Hz, 1H), 5.47 (s, 2H), 5.35 (s, 2H), 4.79 (d, J = 5.49 Hz, 2H). 13C NMR (91 MHz, DMSO-d6) δ 160.8, 156.8, 155.9, 150.1, 148.8, 147.5, 138.3, 136.7, 131.4, 125.7, 122.0, 121.3, 109.3, 98.2, 52.0, 35.7. ESI-HRMS (TOF) m/z: 322.1426 (calcd for C16H15N7O [M + H]+, 322.1416). N4-((5-Methyloxazol-2-yl)methyl)-5-(pyridin-2-ylmethyl)-5Hpyrrolo[3,2-d]pyrimidine-2,4-diamine (37). 1H NMR (360 MHz, DMSO-d6) δ 8.42 (d, J = 3.66 Hz, 1H), 8.01 (t, J = 5.67 Hz, 1H), 7.81 (t, J = 7.43 Hz, 1H), 7.43 (d, J = 2.93 Hz, 1H), 7.25−7.36 (m, 2H), 6.74 (s, 1H), 5.98 (d, J = 2.93 Hz, 1H), 5.47 (s, 2H), 5.34 (s, 2H), 4.72 (d, J = 5.49 Hz, 2H), 2.23 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.1, 158.5, 157.6, 151.7, 150.5, 149.2, 148.9, 138.4, 133.0, 123.7, 123.0, 111.0, 99.9, 53.7, 37.4, 11.0. ESI-HRMS (TOF) m/z: 336.1575 (calcd for C17H17N7O [M + H]+, 336.1572). N4-((4-Methyloxazol-2-yl)methyl)-5-(pyridin-2-ylmethyl)-5Hpyrrolo[3,2-d]pyrimidine-2,4-diamine (38). 1H NMR (360 MHz, DMSO-d6) δ 8.43 (d, J = 4.76 Hz, 1H), 8.03 (t, J = 5.49 Hz, 1H), 7.80 (dt, J = 1.83, 7.68 Hz, 1H), 7.67 (d, J = 1.46 Hz, 1H), 7.43 (d, J = 2.93 Hz, 1H), 7.27−7.36 (m, 2H), 5.98 (d, J = 2.93 Hz, 1H), 5.47 (s, 2H), 5.35 (s, 2H), 4.73 (d, J = 5.49 Hz, 2H), 2.05 (d, J = 1.10 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 162.0, 158.5, 157.6, 151.8, 150.5, 149.2, 138.4, 136.1, 135.2, 133.1, 123.6, 123.0, 111.0, 99.9, 53.8, 37.4, 11.7. ESI-HRMS (TOF) m/z: 336.1573 (calcd for C17H17N7O [M + H]+, 336.1572). 5-(Pyridin-2-ylmethyl)-N4-(thiazol-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (39). 1H NMR (360 MHz, DMSOd6) δ 8.44 (d, J = 4.76 Hz, 1H), 8.12 (t, J = 5.85 Hz, 1H), 7.78 (t, J = 7.73 Hz, 1H), 7.71 (d, J = 3.21 Hz, 1H), 7.52 (d, J = 3.29 Hz, 1H), 7.43 (d, J = 3.29 Hz, 1H), 7.31 (dd, J = 4.94, 6.77 Hz, 1H), 7.14 (d, J = 7.68 Hz, 1H), 6.01 (d, J = 2.93 Hz, 1H), 5.53 (s, 2H), 5.41 (s, 2H), 4.94 (d, J = 5.49 Hz, 2H). 13C NMR (91 MHz, DMSO-d6) δ 169.7, 156.9, 156.3, 150.4, 147.7, 148.7, 141.0, 136.7, 131.8, 122.0, 120.9, 118.6, 109.3, 98.4, 52.1, 40.3. ESI-HRMS (TOF) m/z: 338.1191 (calcd for C16H15N7S [M + H]+, 338.1188). N4-((5-Methylthiazol-2-yl)methyl)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (40). 1H NMR (360 MHz, DMSO-d6) δ 13.09 (br s, 1H), 9.40 (br s, 1H), 8.65 (br d, J = 5.12 Hz, 1H), 8.10 (t, J = 7.32 Hz, 1H), 7.55−7.79 (m, 4H), 7.47 (s, 1H), 7.32 (d, J = 7.68 Hz, 1H), 6.34 (d, J = 2.56 Hz, 2H), 4.95 (br d, J = 4.39 Hz, 2H), 2.36 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 165.6,

153.1, 151.4, 150.2, 145.0, 140.5, 137.3, 135.7, 134.3, 133.2, 123.3, 122.0, 107.6, 94.7, 50.1, 40.3, 10.3. ESI-HRMS (TOF) m/z: 352.1348 (calcd for C17H17N7S [M + H]+, 352.1344). N4-(Isoxazol-3-ylmethyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (41). 13 (175 mg, 0.67 mmol), isoxazol-3-ylmethylamine hydrochloride (136 mg, 1.0 mmol), and DIPEA (173 mg, 1.3 mmol) were dissolved in NMP (2.4 mL) in a 7 mL glass vial. The mixture was stirred at 100 °C for 2 h and then cooled to rt and concentrated in vacuo. It was purified by prep HPLC (stationary phase, RP Vydac Denali C18, 10 μm, 200 g, 5 cm; mobile phase, 0.25% NH4OAc solution in water, CH3OH), and the desired fractions were collected and concentrated in vacuo. The product was triturated in CH3CN, isolated by filtration, and dried under vacuum to afford the title compound as a white solid. 1H NMR (360 MHz, DMSO-d6) δ 8.78 (d, J = 1.46 Hz, 1H), 8.41 (d, J = 4.91 Hz, 1H), 7.90 (t, J = 5.50 Hz, 1H), 7.79 (t, J = 7.55 Hz, 1H), 7.41 (d, J = 2.93 Hz, 1H), 7.31 (dd, J = 4.94, 7.50 Hz, 1H), 7.19 (d, J = 7.68 Hz, 1H), 6.38 (d, J = 1.46 Hz, 1H), 5.99 (d, J = 2.93 Hz, 1H), 5.48 (s, 2H), 5.41 (s, 2H), 4.70 (d, J = 5.85 Hz, 2H). 13C NMR (91 MHz, DMSO-d6) δ 160.7, 158.4, 157.0, 156.3, 150.2, 149.1, 147.6, 136.8, 131.5, 122.1, 121.2, 109.4, 103.4, 98.4, 52.2, 34.1. ESI-HRMS (TOF) m/z: 322.1422 (calcd for C16H15N7O [M + H]+, 322.1416). N4-((5-Methylisoxazol-3-yl)methyl)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (42). 1H NMR (360 MHz, DMSO-d6) δ 8.41 (br d, J = 4.39 Hz, 1H), 8.17 (br s, 1H), 7.80 (br t, J = 7.14 Hz, 1H), 7.48 (d, J = 2.56 Hz, 1H), 7.28−7.38 (m, 1H), 7.21 (br d, J = 7.68 Hz, 1H), 6.06 (d, J = 2.56 Hz, 1H), 5.96 (s, 1H), 5.89 (br s, 2H), 5.52 (s, 2H), 4.65 (br d, J = 4.76 Hz, 2H), 2.33 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.9, 161.4, 155.9, 155.7, 149.4, 147.6, 146.8, 136.8, 132.0, 122.1, 121.2, 109.0, 100.3, 97.4, 52.0, 34.2, 10.6. ESI-HRMS (TOF) m/z: 336.1575 (calcd for C17H17N7O [M + H]+, 336.1572). N 4 -((5-Cyclopropylisoxazol-3-yl)methyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (43). 1H NMR (360 MHz, DMSO-d6) δ 8.42 (d, J = 4.76 Hz, 1H), 7.74− 7.82 (m, 2H), 7.41 (d, J = 2.85 Hz, 1H), 7.33 (t, J = 5.99 Hz, 1H), 7.15 (d, J = 7.68 Hz, 1H), 5.99 (d, J = 2.93 Hz, 1H), 5.89 (s, 1H), 5.49 (s, 2H), 5.41 (s, 2H), 4.59 (d, J = 5.85 Hz, 2H), 2.00−2.12 (m, 1H), 0.96−1.06 (m, 2H), 0.75−0.84 (m, 2H). 13C NMR (91 MHz, DMSOd6) δ 172.7, 161.5, 156.8, 156.1, 150.0, 148.8, 147.4, 147.4, 136.6, 131.3, 121.8, 120.9, 109.1, 98.2, 97.3, 51.9, 33.9, 6.8, 6.3. ESI-HRMS (TOF) m/z: 362.1729 (calcd for C19H19N7O [M + H]+, 362.1729). N4-((5-Isopropylisoxazol-3-yl)methyl)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (44). 1H NMR (360 MHz, DMSO-d6) δ 8.38−8.42 (m, 1H), 7.73−7.82 (m, 2H), 7.42 (d, J = 3.20 Hz, 1H), 7.31 (t, J = 5.90 Hz, 1H), 7.14 (d, J = 8.05 Hz, 1H), 5.99 (d, J = 2.93 Hz, 1H), 5.93 (s, 1H), 5.50 (s, 2H), 5.42 (s, 2H), 4.63 (d, J = 5.49 Hz, 2H), 3.01 (spt, J = 6.95 Hz, 1H), 1.18 (d, J = 6.95 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 178.2, 163.0, 158.6, 157.9, 151.8, 150.6, 149.1, 138.3, 133.1, 123.6, 122.6, 110.9, 100.0, 99.3, 53.7, 35.7, 26.9, 21.1. ESI-HRMS (TOF) m/z: 364.1891 (calcd for C19H21N7O [M + H]+, 364.1886). N4-((3-Methylisoxazol-5-yl)methyl)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (45). 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 5.01 Hz, 1H), 7.89 (t, J = 5.67 Hz, 1H), 7.80 (t, J = 7.71 Hz, 1H), 7.42 (d, J = 3.11 Hz, 1H), 7.34 (t, J = 6.19 Hz, 1H), 7.19 (d, J = 7.92 Hz, 1H), 6.00 (d, J = 3.08 Hz, 1H), 5.95 (s, 1H), 5.50 (s, 2H), 5.37 (s, 2H), 4.71 (d, J = 5.50 Hz, 2H), 2.16 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 171.4, 159.8, 158.5, 157.9, 151.9, 149.3, 150.3, 138.4, 133.2, 123.7, 122.7, 111.0, 103.0, 100.0, 53.7, 36.1, 11.4. ESI-HRMS (TOF) m/z: 336.1577 (calcd for C17H17N7O [M + H]+, 336.1572). 5-((5,6-Dimethoxypyridin-2-yl)methyl)-N4-((5-methylisoxazol-3-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (46). 1 H NMR (360 MHz, DMSO-d6) δ 7.35 (d, J = 2.93 Hz, 1H), 7.20 (d, J = 7.68 Hz, 1H), 6.87 (t, J = 5.85 Hz, 1H), 6.54 (d, J = 8.05 Hz, 1H), 5.99 (d, J = 2.93 Hz, 1H), 5.86 (s, 1H), 5.39 (s, 2H), 5.35 (s, 2H), 4.61 (d, J = 5.85 Hz, 2H), 3.71 (d, J = 2.93 Hz, 6H), 2.31 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.6, 161.6, 156.7, 151.9, 150.1, 148.5, 143.4, 141.7, 131.6, 117.5, 113.0, 109.0, 100.0, 98.0, 54.2, 51.5, 51.2, 6148

DOI: 10.1021/acs.jmedchem.7b00365 J. Med. Chem. 2017, 60, 6137−6151

Journal of Medicinal Chemistry

Article

4-((5-Methylisoxazol-3-yl)methoxy)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidin-2-amine (54). 1H NMR (360 MHz, DMSO-d6) δ 8.47 (qd, J = 0.96, 4.80 Hz, 1H), 7.65 (dt, J = 1.83, 7.68 Hz, 1H), 7.53 (d, J = 2.93 Hz, 1H), 7.24 (ddd, J = 1.10, 4.76, 7.68 Hz, 1H), 6.71 (d, J = 8.05 Hz, 1H), 6.14 (d, J = 2.93 Hz, 1H), 5.92 (s, 2H), 5.85 (d, J = 1.10 Hz, 1H), 5.47 (s, 2H), 5.37 (s, 2H), 2.35 (d, J = 0.73 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 168.4, 158.9, 156.7, 156.7, 153.8, 152.3, 147.8, 135.8, 133.3, 121.1, 119.0, 108.3, 100.0, 98.2, 56.6, 52.2, 10.5. ESI-HRMS (TOF) m/z: 337.1416 [M + H]+ (calcd for C17H16N6O2: 337.1413). 4-((5-Methyloxazol-2-yl)methoxy)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidin-2-amine (55). 1H NMR (360 MHz, DMSO-d6) δ 8.43 (br d, J = 4.39 Hz, 1H), 7.62 (t, J = 7.83 Hz, 1H), 7.51−7.54 (m, 1H), 7.18−7.25 (m, 1H), 6.82 (s, 1H), 6.74 (d, J = 7.68 Hz, 1H), 6.13 (d, J = 2.20 Hz, 1H), 5.89 (s, 2H), 5.43 (s, 2H), 5.39 (s, 2H), 2.24 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.7, 156.5, 156.5, 156.4, 153.7, 152.4, 148.5, 147.8, 135.6, 133.5, 121.9, 121.2, 119.2, 98.2, 56.8, 52.3, 9.4. ESI-HRMS (TOF) m/z: 337.1413 (calcd for C17H16N6O2 [M + H]+, 337.1413). 4-((5-Methylthiazol-2-yl)methoxy)-5-(pyridin-2-ylmethyl)5H-pyrrolo[3,2-d]pyrimidin-2-amine (56). 1H NMR (360 MHz, DMSO-d6) δ 8.46 (d, J = 4.76 Hz, 1H), 7.63 (dt, J = 1.46, 7.68 Hz, 1H), 7.52 (d, J = 2.93 Hz, 1H), 7.43 (d, J = 1.10 Hz, 1H), 7.22 (dd, J = 4.94, 7.14 Hz, 1H), 6.71 (d, J = 7.68 Hz, 1H), 6.15 (d, J = 3.29 Hz, 1H), 5.92 (s, 2H), 5.58 (s, 2H), 5.49 (s, 2H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 163.7, 158.3, 158.3, 155.2, 154.2, 149.5, 140.5, 137.4, 135.1, 135.5, 122.8, 120.8, 110.1, 100.0, 63.5, 53.9, 11.8. ESI-HRMS (TOF) m/z: 353.1183 [M + H]+ (calcd for C17H16N6OS: 353.1184). 4-((5-Methyl-1,2,4-oxadiazol-3-yl)methoxy)-5-(pyridin-2-ylmethyl)-5H-pyrrolo[3,2-d]pyrimidin-2-amine (57). 1H NMR (360 MHz, DMSO-d6) δ 8.43 (br d, J = 4.03 Hz, 1H), 7.64 (br t, J = 7.68 Hz, 1H), 7.53 (d, J = 2.93 Hz, 1H), 7.16−7.28 (m, 1H), 6.84 (d, J = 7.68 Hz, 1H), 6.13 (d, J = 2.93 Hz, 1H), 5.89 (s, 2H), 5.51 (br s, 2H), 5.46 (br s, 2H), 2.54−2.61 (m, 3H). 13C NMR (91 MHz, DMSO-d6) δ 176.0, 165.1, 156.5, 156.3, 153.5, 152.3, 147.6, 135.5, 133.3, 121.0, 119.3, 108.2, 98.1, 55.6, 52.1, 10.6. ESI-HRMS (TOF) m/z: 338.1364 (calcd for C16H15N7O2 [M + H]+, 338.1365). 5-((3-Methoxypyridin-2-yl)methyl)-4-((5-methyloxazol-2-yl)methoxy)-5H-pyrrolo[3,2-d]pyrimidin-2-amine (58). 1H NMR (360 MHz, DMSO-d6) δ 7.87 (dd, J = 1.10, 4.39 Hz, 1H), 7.37 (d, J = 2.93 Hz, 1H), 7.33 (dd, J = 1.10, 8.42 Hz, 1H), 7.21 (dd, J = 4.76, 8.05 Hz, 1H), 6.80 (d, J = 1.10 Hz, 1H), 6.07 (d, J = 2.93 Hz, 1H), 5.80 (s, 2H), 5.45 (s, 2H), 5.32 (s, 2H), 3.77 (s, 3H), 2.22 (d, J = 1.10 Hz, 3H). 13C NMR (91 MHz, DMSO-d6) δ 156.4, 156.3, 153.7, 152.0, 150.9, 148.4, 144.7, 138.8, 133.6, 122.0, 121.8, 116.2, 108.9, 97.5, 56.6, 54.2, 47.2, 9.3. ESI-HRMS (TOF) m/z: 367.1520 (calcd for C18H18N6O3 [M + H]+, 367.1518). 5-((3-Methoxypyridin-2-yl)methyl)-4-((5-methylisoxazol-3yl)methoxy)-5H-pyrrolo[3,2-d]pyrimidin-2-amine (59). 1H NMR (360 MHz, DMSO-d6) δ 7.89 (d, J = 4.39 Hz, 1H), 7.32− 7.43 (m, 2H), 7.24 (dd, J = 4.76, 8.05 Hz, 1H), 6.06 (d, J = 2.56 Hz, 1H), 5.82 (br s, 2H), 5.77 (br s, 1H), 5.47 (s, 2H), 5.30 (s, 2H), 3.79 (s, 3H), 2.35 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 168.4, 159.0, 156.4, 153.8, 152.0, 151.0, 144.8, 138.9, 133.5, 122.1, 116.3, 109.0, 99.9, 97.6, 56.5, 54.2, 47.3, 10.6. ESI-HRMS (TOF) m/z: 367.1519 (calcd for C18H18N6O3 [M + H]+, 367.1518).

34.0, 10.3. ESI-HRMS (TOF) m/z: 396.1787 (calcd for C19H21N7O3 [M + H]+, 396.1784). 5-((6-Methoxypyridin-2-yl)methyl)-N4-((5-methylisoxazol-3yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (47). 1H NMR (400 MHz, DMSO-d6) δ 7.58−7.65 (m, J = 8.10 Hz, 1H), 7.36 (d, J = 2.86 Hz, 1H), 6.90 (t, J = 5.83 Hz, 1H), 6.69 (d, J = 8.36 Hz, 1H), 6.50 (d, J = 7.26 Hz, 1H), 5.99−6.03 (m, 1H), 5.83 (s, 1H), 5.44 (s, 2H), 5.36 (s, 2H), 4.59 (d, J = 5.72 Hz, 2H), 3.71 (s, 3H), 2.31 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.7, 162.1, 161.8, 157.0, 154.5, 150.4, 148.8, 139.1, 132.0, 112.8, 109.4, 108.2, 100.2, 98.5, 51.9, 51.9, 34.2, 10.6. ESI-HRMS (TOF) m/z: 366.1678 (calcd for C18H19N7O2 [M + H]+, 366.1678). 5-((5-Fluoropyridin-2-yl)methyl)-N4-((5-methylisoxazol-3-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (48). 1H NMR (360 MHz, DMSO-d6) δ 8.42 (d, J = 2.93 Hz, 1H), 7.70 (dt, J = 2.93, 8.78 Hz, 1H), 7.39 (d, J = 2.93 Hz, 1H), 7.31 (t, J = 5.67 Hz, 1H), 7.09 (dd, J = 4.57, 8.60 Hz, 1H), 6.00 (d, J = 2.93 Hz, 1H), 5.88 (s, 1H), 5.52 (s, 2H), 5.41 (s, 2H), 4.59 (d, J = 5.85 Hz, 2H), 2.32 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.6, 161.6, 156.9, 155.7, 152.7, 150.2, 148.6, 135.6, 131.5, 123.3, 121.9, 108.9, 100.1, 98.3, 51.2, 33.8, 10.4. ESI-HRMS (TOF) m/z: 354.1480 (calcd for C17H16FN7O [M + H]+, 354.1478). 5-((3-Fluoropyridin-2-yl)methyl)-N4-((5-methylisoxazol-3-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (49). 1H NMR (360 MHz, DMSO-d6) δ 8.22 (d, J = 4.76 Hz, 1H), 7.78 (t, J = 9.25 Hz, 1H), 7.63 (t, J = 5.58 Hz, 1H), 7.47 (td, J = 4.35, 8.51 Hz, 1H), 7.26 (d, J = 3.23 Hz, 1H), 6.06 (s, 1H), 5.97 (d, J = 2.93 Hz, 1H), 5.58 (d, J = 1.83 Hz, 2H), 5.39 (s, 2H), 4.63 (d, J = 5.49 Hz, 2H), 2.34 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.8, 161.7, 156.9, 150.0, 148.9, 143.8, 143.5, 131.6, 124.5, 123.3, 123.2, 109.6, 100.5, 98.4, 46.7, 34.1, 10.6. ESI-HRMS (TOF) m/z: 354.1479 (calcd for C17H16FN7O [M + H]+, 354.1478). N4-((5-Methylisoxazol-3-yl)methyl)-5-((5-(trifluoromethyl)pyridin-2-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (50). 1H NMR (360 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.17 (dd, J = 2.20, 8.42 Hz, 1H), 7.41 (d, J = 2.93 Hz, 1H), 7.10 (t, J = 5.67 Hz, 1H), 7.02 (d, J = 8.25 Hz, 1H), 6.05 (d, J = 2.93 Hz, 1H), 5.74 (s, 1H), 5.69 (s, 2H), 5.43 (s, 2H), 4.56 (d, J = 5.49 Hz, 2H), 2.28 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.5, 161.6, 161.2, 157.0, 150.5, 148.6, 144.4, 144.4, 133.8, 133.7, 131.9, 120.0, 109.0, 99.9, 98.7, 51.7, 33.7, 10.3. ESI-HRMS (TOF) m/z: 404.1450 (calcd for C18H16F3N7O [M + H]+, 404.1446). N4-((5-Methylisoxazol-3-yl)methyl)-5-((4-(trifluoromethyl)pyridin-2-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (51). 1H NMR (360 MHz, DMSO-d6) δ 8.72 (d, J = 5.12 Hz, 1H), 7.72 (d, J = 5.12 Hz, 1H), 7.30−7.45 (m, 3H), 6.03 (d, J = 3.29 Hz, 1H), 5.83 (s, 1H), 5.65 (s, 2H), 5.43 (s, 2H), 4.60 (d, J = 5.85 Hz, 2H), 2.31 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.5, 161.6, 158.2, 156.9, 150.3, 149.4, 148.6, 131.7, 117.3, 117.3, 115.9, 115.8, 109.0, 99.9, 98.6, 51.5, 33.8, 10.3. ESI-HRMS (TOF) m/z: 404.1451 (calcd for C18H16F3N7O [M + H]+, 404.1446). 5-((3-Chloropyridin-2-yl)methyl)-N4-((5-methylisoxazol-3yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (52). 1H NMR (360 MHz, DMSO-d6) δ 8.32 (d, J = 4.76 Hz, 1H), 8.00 (d, J = 8.05 Hz, 1H), 7.71 (br t, J = 5.49 Hz, 1H), 7.42 (dd, J = 4.57, 8.23 Hz, 1H), 7.30 (d, J = 2.93 Hz, 1H), 6.03 (s, 1H), 5.94−6.00 (m, 1H), 5.62 (s, 2H), 5.44 (s, 2H), 4.62 (d, J = 5.49 Hz, 2H), 2.34 (s, 3H). 13C NMR (91 MHz, DMSO-d6) δ 167.6, 161.6, 156.7, 152.0, 149.6, 148.7, 146.0, 136.9, 131.7, 128.7, 123.7, 109.7, 100.3, 98.0, 49.4, 33.9, 10.4. ESI-HRMS (TOF) m/z: 370.1183 (calcd for C17H16ClN7O [M + H]+, 370.1183). 5-((4-Chloropyridin-2-yl)methyl)-N4-((5-methylisoxazol-3yl)methyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (53). 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 5.50 Hz, 1H), 7.48 (dd, J = 1.87, 5.39 Hz, 1H), 7.36−7.45 (m, 2H), 7.17−7.23 (m, 1H), 6.01 (d, J = 3.08 Hz, 1H), 5.89 (s, 1H), 5.52 (br s, 2H), 5.38 (br s, 2H), 4.60 (d, J = 5.72 Hz, 2H), 2.33 (s, 3H). 13C NMR (91 MHz, DMSOd6) δ 167.7, 161.6, 158.3, 157.0, 150.3, 149.2, 148.7, 142.8, 131.6, 121.9, 120.6, 109.0, 100.1, 98.5, 51.4, 33.9, 10.5. ESI-HRMS (TOF) m/z: 370.1183 (calcd for C17H16ClN7O [M + H]+, 370.1183).



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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00365. Molecular formula strings and some data (CSV) Experimental details, docking methods, biological assay details, and NMR spectra of 54 (PDF) 6149

DOI: 10.1021/acs.jmedchem.7b00365 J. Med. Chem. 2017, 60, 6137−6151

Journal of Medicinal Chemistry



Article

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AUTHOR INFORMATION

Corresponding Authors

*D.C.M.: phone, +32 6414 1019; e-mail, [email protected]. com. *F.H.: phone, +32 6414 1644; e-mail, [email protected]. ORCID

David C. McGowan: 0000-0002-3759-6771 Notes

The authors declare no competing financial interest. Authors will release the atomic coordinates and experimental data upon article publication.



ACKNOWLEDGMENTS Thanks are extended to Eddy De Wilde, Kristien Raeymaekers, Michel Carpentier, Alex De Groot, Hilde Verheyen, and Alberto Fontana for analysis and purification, and Monicah Otieno for bioanalysis.



ABBREVIATIONS USED HBV, hepatitis B virus; TLR, Toll-like receptor; IFN, interferon; TNF, tumor necrosis factor; NF-κB, nuclear factor κB; pDC, plasmacytoid dentritic cell; ISG, interferon stimulated gene; PBMC, peripheral blood mononuclear cell



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DOI: 10.1021/acs.jmedchem.7b00365 J. Med. Chem. 2017, 60, 6137−6151

Journal of Medicinal Chemistry

Article

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DOI: 10.1021/acs.jmedchem.7b00365 J. Med. Chem. 2017, 60, 6137−6151